Endotoxin (or lipopolysaccharide; LPS) is a main cause of lethal septic shock. Its toxicity is primarily mediated by the host overproduction of tumor necrosis factor alpha (TNF-(). Identifying agents that neutralize endotoxin and inhibit TNF-( production represents, therefore, a logical approach to prevention or treatment of the disease. To this end, we have used two independent assays (Limulus assay and TNF-( bioassay) to quantify the endotoxin-neutralizing activity of potential anti-LPS agents. Here we report a nonionic detergent Triton X-114 and a novel synthetic antibacterial peptide corresponding to the 33-mer at the N-terminus of human lactoferrin as new anti-LPS agents. The peptide inhibited Limulus coagulation and induction of TNF-a secretion by 4 different types of LPS and lipid A with equivalent or higher potency than polymyxin B. The anti-LPS potency of Triton X-114 was comparably lower than that of the peptide. The peptide neutralized LPS by binding to the lipid A portion. The first 6 residues (including 4 cationic residues) at the N-terminus of the peptide were crucial for the anti-LPS activity. The anti-LPS activity of Triton X-114 occurred above its critical micellization concentration (0.3 mM). Premixing of E.coli LPS (125 nanogram) with the peptide (2.5 microgram) or Triton X-114 (100 microgram) completely prevented the lethality resulting from endotoxic shock in the galactosamine-sensitized mouse model. Combination of the peptide with Triton X-114 gave a synergistic, significant protection even when they were injected intravenously 10 min after intraperitoneal injection of LPS. The protection was correlated with the reduction of the mouse serum TNF-a level. These results demonstrate the potential use of the lactoferrin-derived peptide and Triton X-114 as novel therapeutic agents against endotoxic shock.